Biological Electron Microscopy Theory, Techniques, and Troubleshooting

Biological Electron Microscopy Theory, Techniques, and Troubleshooting

MICHAEL J. DYKSTRA College of Veterinary Medicine North Carolina State University Raleigh, North Carolina

PLENUM PRESS • NEW YORK AND LONDON

Library of Congress Cataloging in Publication Data

Dykstra, Michael J. Biological electron microscopy: theory, techniques, and troubleshooting / Michael

J. Dykstra. p. cm.

Includes bibliographical references (p. ) and index. ISBN-13: 978-1-4684-0012-0 e-ISBN-13: 978-1-4684-0010-6 DOl: 10.1007/978-1-4684-0010-6 1. Electron microscopy. 2. Scanning electron microscopy. 3. Transmission electron

microscopes. I. Title. QH212.E4D95 1992 92-34102 578'.45-dc20 CIP

ISBN-13: 978-1-4684-0012-0

© 1992 Michael J. Dykstra Softcover reprint ofthe hardcover 1st edition 1992

Plenum Press is a division of Plenum Publishing Corporation 233 Spring Street, New York, N.Y. 10013

All rights reserved

No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written permission from the Publisher

To the two Susans, for patience

Preface

Electron microscopy is frequently portrayed as a technically demanding discipline operating largely in the sphere of "black boxes" and governed by many absolute laws of procedure. At the introductory level, this description does the discipline and the student a disservice. The instrumentation we use is complex but ultimately understand­able and, more important, reparable. The chemical and physical processes underlying the procedures we employ for preparing tissues and cells are not totally elucidated, but enough information is available to allow investigators to make reasonable choices concerning the best techniques to apply to their particular problems. There are count­less specialized techniques in the field of electron microscopy that require the acquisi­tion of specialized knowledge, particularly for the interpretation of results (energy­dispersive spectroscopy comes immediately to mind), but most laboratories with equip­ment to utilize these approaches have specialists to help the casual user.

There are several books available that deal with biological electron microscopy. Some (e.g., Meek, 1976) are extremely good in regard to instrumentation, whereas others are heavily skewed toward specimen preparation (e.g., Hayat, 1989). Some, such as that by Wischnitzer (1981), cover both specimen preparation and instrumenta­tion in almost sufficient detail for an introductory course. Unfortunately, most single­volume electron microscopy texts were written prior to 1983 and are thus quite dated. There are also several series of books (see the Hayat series and the Glauert series in Appendix B) containing quite complete and up-to-date information on many subjects, but these are too expensive and too extensive to be considered as textbooks. There are also a number of laboratory procedure manuals available, but these consistently state that the limited techniques they list are the only ones that can be used. This is clearly not the case.

Most of my contemporaries were taught fear, rather than respect, for the instru­mentation of electron microscopy. In addition, we were generally taught that there was only one right way to fix and embed a given class of organisms, only one way to properly break a glass knife, only one side of a grid to use for section retrieval, and

vii

viii PREFACE

only one way to properly post-stain grids. After going to various meetings for 20 years and hearing all the different types of approaches utilized to obtain publishable ultra­structural work, I see clearly that the system is much more flexible than our training had suggested.

This textbook is offered to help a beginning student see how things work in electron microscopy. It presents the principles of fixation and specimen preparation, showing the breadth of approaches that are practical. It gives students sufficient infor­mation about basic specimen preparation to make cogent decisions for their own projects after consulting the relevant literature for their own group of organisms or tissues. There are still black boxes in the discipline, but I will try to minimize them.

A major component of this book is suggested by the subtitle: Theory, Techniques, and Troubleshooting. Too many of my generation were given in their training little theory beyond optical theory; too little information about techniques, except the ones used in their specific laboratory; and almost nothing about troubleshooting problems, particularly in regard to instrumentation. Clearly, things do go wrong, and with alarm­ing regUlarity.

In a discipline with so many varied approaches from which to choose, teaching how to approach a problem logically and how to correct a problem that presents itself probably represents the highest aim of a course in electron microscopy.

This book is intended for a one-semester course that covers all the basic ap­proaches utilized in transmission and scanning electron microscopy. Its arrangement is somewhat unusual in being more or less chronological, so that the student can be reading about the techniques as they are employed in class.

Finally, one of the major purposes of this text is to provide more up-to-date coverage of the two areas that have revolutionized electron microscopy over the last 8-10 years: cryotechniques and immunolabeling. When glutaraldehyde was introduced by Sabatini et al. in 1963, chemical fixation more or less reached its apex. Until the advent of easily reproducible cryotechniques, no further advances took place in speci­men preparation for transmission electron microscopy. By the late 1970s, the discipline was perceived by many as a fairly passe descriptive science with little innovation except in the area of high-voltage electron microscopy. Fortunately, in the 1980s there was improved instrumentation for cryo work, and the coupling of immunology and electron microscopy through immunogold and immunoperoxidase techniques has brought our discipline back on line. The power of current techniques to utilize molecu­lar approaches to probe the location of various cellular products and activities has rekindled interest in the truly dynamic aspects of cellular behavior.

A student who masters the concepts in this text will be capable, with continued practice in technical skills, to utilize electron microscopy techniques productively in his or her research. It is my hope that this text will give a firm foundation on which the student can build knowledge of cellular structure and behavior far beyond the scope of this work.

Hayat. M.A. 1989. Fixation for electron microscopy. Academic Press. New York. Meek, G.A. 1976. Practical electron microscopy for biologists, 2nd ed. John Wiley & Sons, New York. Wi schnitzer, S. 1981. Introduction to electron microscopy, 3rd ed. Pergamon Press, New York.

Contents

Introduction 1

Chapter 1

Specimen Preparation for Transmission Electron Microscopy .. . . . . . . . . . 5 I. Fixatives ................................................. 5

A. Purpose; Killing versus Fixing ............................ 5 B. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

II. Buffers ................................................... 24 A. Molarity/Molality/Osmolarity/Tonicity . . . . . . . . . . . . . . . . . . . . . . 24 B. Purpose ............................................... 29 C. Types, Characteristics, and Uses of Buffers. . . . . . . . . . . . . . . . . . 30

III. Dehydration ................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 A. Purpose ............................................... 32 B. Agents ................................................ 32

IV. Embedding Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 A. Ideal Qualities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 B. Classes and Characteristics of Resins ....................... 34 C. Embedding Mold Types........ ............. ........ .... . 38 D. Agar Embedment........................... ............ 38

V. Examination of Four Tissues Prepared with a Variety of Fixatives and Buffers ............................................... 39

VI. A Quasi-Universal Fixation, Dehydration, and Embedment Schedule Successfully Used on Organisms from All Five Kingdoms of Life .. 68

References ................................................... 77

Chapter 2

Ultramicrotomy .................................................. 79 I. Ultramicrotomes ........................................... 79

ix

x CONTENTS

A. Purpose ............................................... 79 B. Design ................................................ 79 C. History. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

II. Knives ................................................... 82 A. History. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 B. Glass Knive Manufacturing ............................... 83 C. Section Handling ....................................... 85 D. Knife Storage .......................................... 86 E. Diamond Knives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

III. Block Trimming ........................................... 87 A. Trimming Procedures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 B. Block-Trimming Tools ................................... 90

IV. Sectioning Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 A. Working Area. . . .. . . . . . . .. .. .. . . . . ..... ... ... ... .. .. ... 91 B. Sectioning Procedures ................................... 93

v. Eight Commonly Encountered Sectioning Problems .............. 97 References ............................... '.................... 101

Chapter 3

Support Films 103

I. Purpose .................................................. 103 II. Types .................................................... 103

A. Nitrocellulose .......................................... 103 B. Formvar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 C. Carbon. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

III. Methods.................................................. 104 A. Droplet ............................................... 105 B. Slide Stripping ......................................... 105 C. Holey Films ........................................... 111 D. Carbon Films .......................................... 112

References ................................................... 112

Chapter 4

Transmission Electron Microscopy .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 113

I. Historical Review of Microscopy (1590-1990s) ................. 113 II. Theory of Electron Optics ................................... 115

A. Light Microscopes versus the TEM ........................ 115 B. Resolution ............................................. 115 C. Electron Lenses ........................................ 117 D. Properties of Electron Lenses ............................. 118

CONTENTS xi

III. Four Aspects of Image Formation ............................. 127 A. Absorption ............................................ 127 B. Interference ............................................ 127 C. Diffraction .......................................... . . . 127 D. Scattering ............................................. 128

IV. General TEM Features ...................................... 128 A. Operating Voltage. . . .. . . . . . . . . . . . .. . .. . . . . . . . . . . . . . . . . .. 128 B. Resolution ............................................. 128 C. Magnification .......................................... 129 D. High Vacuum, Electronic, Magnetic, and Physical Stability .... 129

V. Parts of the Electron Microscope: Functional Aspects. . . . . . . . . . . . . 129 A. The Electron Gun ....................................... 131 B. Condenser Lens System .............. . . . . . . . . . . . . . . . . . . .. 134 C. Deflector Coils ......................................... 135 D. Objective Lens ......................................... 135 E. Diffraction Lens ........................................ 138 F. Projector System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 G. Camera Systems ........................................ 138 H. Specimen Holders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139 I. Viewing System ........................................ 139 1. Detectors .............................................. 141

VI. Operation of the TEM: Decision Making ....................... 141 A. Accelerating Voltage .................................... 141 B. Choice of Beam Current and Bias ......................... 141 C. Condenser Settings ...................................... 142 D. Objective Settings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 142 E. Alignment ............................................. 142 F. Taking a Photograph . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 G. Specimen Radiation Dose ................................ 148 H. Microscope Calibration .................................. 149

References ................................................... 150

Chapter 5

Vacuum Systems 151

I. Types of Gauges ........................................... 151 A. Direct Reading ......................................... 151 B. Indirect Gauges. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 152

II. Vacuum Pumps ............................................ 155 A. Types of Pumping Systems ............................... 155 B. Mechanical/Rotary Vane Pumps ........................... 155 C. Diffusion Pumps. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 D. Turbomolecular Pumps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159

xii CONTENTS

E. Sputter Ion (Ion Getter) Pumps ............................ 161 F. Cryopumps and "Cold Fingers" ........................... 162

III. Sequential Operation of a Complete Vacuum System to Achieve High Vacuum ............................................. 162

IV. Lubrication of Vacuum Seals and Leak Detection ................ 164 References ................................................... 165

Chapter 6

Staining Methods for Semithins and Ultrathins 167

I. Semithin Section Staining ................................... 167 A. Toluidine Blue-O ....................................... 168 B. Toluidine Blue-O and Acid Fuchsin ........................ 169 C. Basic Fuchsin/Methylene Blue ............................ 170 D. Methylene Blue ........................................ 171 E. Periodic Acid/Schiff's Reagent. ..... .... .. .. .. ...... .. . ... 171

II. Ultrathin Section Staining ................................... 171 A. Purpose ............................................... 171 B. En Bloc versus Post-Staining. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 172 C. Commonly Used Post-Stains .............................. 173 D. Microwave Staining ..................................... 180 E. Dark-Field Imaging without Staining ....................... 180

References .................................................. 181

Chapter 7

Photography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 183

I. Emulsion Composition ...................................... 184 II. Film Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

III. Producing a Latent Image ................................... 187 IV. Film Processing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

A. Developer ............................................. 188 B. Stop Baths. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 C. Fixer ................................................. 190

V. Development Controls ...................................... 190 A. Time ................................................. 190 B. Temperature ........................................... 190 C. Agitation .............................................. 191 D. Developer Choice ....................................... 191

VI. Paper Types ............................................... 191 VII. Keeping Properties of Chemicals and Precautions ................ 193

VIII. Sharpness................................................. 193

CONTENTS xiii

IX. Films Used in the Electron Microscopy Laboratory. . . . . . . . . . . . . . . 194 A. Negative-Release Films .................................. 194 B. Positive-Release Films ................................... 195

X. Copy Work ............................................... 198 A. Films ................................................. 198 B. Improving Copy-Stand Images ............................ 199

XI. Types of Enlargers ......................................... 204 A. Diffusion Enlargers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 204 B. Condenser Enlargers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 205 C. Point Light Source Enlargers . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 205 D. LogEtronics Enlargers ................................... 206

XII. Viewing a Print in Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 206 References .................................................... 207

Chapter 8

Replicas, Shadowing, and Negative Staining . . . . . . . . . . . . . . . . . . . . . . . . .. 209

I. Shadowing Casting ......................................... 209 A. Mechanism ............................................ 210 B. Metals Used ........................................... 211 C. Vacuum Evaporators. . . .. .. . . . . .. . . .. . .. . . . . . . . . . .. . . . . .. 211 D. Electrodes ............................................. 214 E. Factors Leading to Fine Grains of Shadowed Metal ........... 215 F. Shadowing Techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 216 G. Sputter Coating. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 216

II. Negative Staining .......................................... 218 A. Mechanism ............................................ 218 B. Methods. . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . . . .. 219

References ................................................... 221

Chapter 9

Scanning Electron Microscopy 223

I. History ................................................... 223 II. The Use of SEM in Biological Research and Medicine ........... 224

III. Principles of the SEM ...................................... 228 IV. Operation of the SEM ...................................... 229 V. Interaction of the Electron Beam and Specimen ................. 230

A. Resolution ............................................. 230 B. Secondary Electrons ..................................... 232 C. Backscattered Electrons .................................. 234 D. Auger Electrons ........................................ 235

xiv CONTENTS

E. Energy-Dispersive Spectroscopy (EDS) ..................... 236 F. Cathodoluminescence .................................... 237

VI. Specimen Preparation ....................................... 237 A. Fixation ............................................... 237 B. Dehydration and Transition Fluids ......................... 238 C. Drying ................................................ 238 D. Mounting Specimens .................................... 240 E. Coating Specimens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 241

VII. Artifacts and Their Correction ................................ 242 VIII. Specialty SEMS: FEG, LV, and ESEM Devices ................. 243 References .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 245

Chapter 10

Cryotechniques 247

I. History ................................................... 248 A. Organismal Period ...................................... 248 B. Mechanistic Period ...................................... 248 C. Cytological Period ...................................... 249

II. Purpose .................................................. 249 III. Cryogens ................................................. 251 IV. Safety Precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 251 V. Freezing Methods .......................................... 252

A. High-Speed Plunging/Immersion .......................... 252 B. Spray Freezing ......................................... 254 C. Jet Freezing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 254 D. High-Pressure Freezing .................................. 255 E. Metal Mirror/Slam Freezing .............................. 257

VI. Uses of Frozen Specimens ................................... 258 A. Cryoultramicrotomy ..................................... 260 B. Cryosubstitution ........................................ 262 C. Freeze-Fracture ......................................... 264 D. Freeze-Drying as Typified by Molecular Distillation. . . . . . . . . .. 267

VII. Artifacts and Their Correction ................................ 270 References .................................................... 272

Chapter 11

High-Voltage Electron Microscopy 275

I. History ................................................... 275 II. Purpose .................................................. 276

III. Functional Aspects of HVEMS ............................... 276 A. Resolution ............................................. 276

CONTENTS xv

B. Radiation Damage ...................................... 277 C. Contrast.. . . . . . . . . . . . . . . .. .. . . .. .. . . . . . . . . . .. . . . . . . . . .. 278

IV. Microscope Construction .................................... 279 V. Sample Preparation ..................................... . . .. 280

VI. Applications .............................................. 280 VII. Intermediate-Voltage Electron Microscopy (IVEM) ............... 281 References .................................................... 283

Chapter 12

Microanalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 285

1. Microanalysis Techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 285 A. Energy-Dispersive Spectroscopy (EDS) ..................... 285 B. Electron Energy Loss Spectroscopy (EELS) ................. 291

References .................................................. 293

Chapter 13

Cytochemistry 295

I. Problems ................................................. 295 II. Specific Reaction Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 297

A. Peroxidase Procedures ................................... 298 B. Lead Capture .......................................... 298 C. Ferritin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 298 D. Colloidal Gold ......................................... 299 E. Ruthenium Red, Alcian Blue, Pyroantimonate ............... 299

III. Examples of Enzyme Cytochemistry . . . . . . . . . . . . . . . . . . . . . . . . . .. 299 A. Peroxidase Methods ..................................... 299 B. Hatchett's Brown Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 300 C. Lead-Capture Methods. . . . . . . . . . . . . . .. . . . . . . . .. . . . . . . . . .. 300

IV. Examples of Nonenzymatic Cytochemistry . . . . . . . . . . . . . . . . . . . . .. 302 A. Cationic Dyes .......................................... 302 B. Polysaccharide Stains .................................... 302 C. Monosaccharide and Disaccharide Stains .................... 305 D. Calcium Staining ....................................... 305

References ................................................... 307

Chapter 14

Immunocytochemistry 309

I. Purpose .................................................. 309 II. Preparative Techniques ...................................... 310

xvi CONTENTS

A. Pre-Embedding Labeling ................................. 310 B. Post-Embedding Procedures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 311 C. Cryoultramicrotomy Technique ............................ 313 D. Negative Staining Procedures ............................. 314

III. Immunoglobulins .......................................... 315 A. Protein A and Protein G Techniques. . . . . . . . . . . . . . . . . . . . . . .. 315 B. Polyclonal and Monoclonal Antibodies ..................... 315

IV. Common Immunolabeling Techniques for Electron Microscopy. . . .. 315 A. Immunoferritin ......................................... 315 B. Immunoperoxidase Techniques ............................ 318 C. Immunogold Techniques ................................. 318

References ................................................... 319

Chapter 15

Autoradiography 321

I. History ................................................... 321 II. Purpose .................................................. 322

III. Theoretical Aspects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 322 A. Detection of Radioactivity ................................ 322 B. Types of Particles ....................................... 322 C. Nuclear Emulsions ...................................... 324 D. Determination of Isotope Dose Level ....................... 325 E. Rules for Autoradiography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 326 F. Light Microscopy Autoradiography ........................ 330

References ................................................... 331

Chapter 16

Computer-Assisted Imaging. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 333

I. Purpose .................................................. 333 II. Resolution and Discrimination ... . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 334

III. Image Processing .......................................... 335 IV. Morphometric Analysis ..................................... 337 V. Stereology ................................................ 337

VI. Computer-Assisted Analysis of Movement ...................... 339 References .................................................... 340

Chapter 17

Scanning Tunneling Microscopy and Its Derivatives 341

CONTENTS

Appendix A

Laboratory Safety

Appendix B

xvii

345

Literature Sources for Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . .. 347

I. Atlases ................................................... 347 II. Journals .................................................. 347

III. Society Publications ........................................ 348 IV. Book Series ............................................... 348 V. NIH Resources for Intermediate Voltage Electron Microscopy

(IVEM) .................................................. 348

Appendix C

Electron Microscopy Equipment and Supplies 349

I. Expendable Supplies and Small Equipment ..................... 349 II. Electron Microscopes ....................................... 350

III. Diamond Knives ........................................... 351 IV. High-Vacuum Pumps ....................................... 352 V. Ultramicrotomes ........................................... 352

VI. Equipment for Cryotechniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 352 VII. Sputter Coaters and Vacuum Evaporators ....................... 353

Index........................................................... 355

Biological Electron Microscopy Theory, Techniques, and Troubleshooting